Synaptosomal Preparations Research Articles

Ex vivo identification of protein–protein interactions involving the dopamine transporter

The dopamine (DA) transporter (DAT) is a key regulator of dopaminergic signaling as it mediates the reuptake of extrasynaptic DA and thereby terminates dopaminergic signaling. Emerging evidence indicates that DAT function is influenced through interactions with other proteins. The current report describes a method to identify such interactions following DAT immunoprecipitation from a rat striatal synaptosomal preparation. This subcellular fraction was selected since DAT function is often determined ex vivo by measuring DA uptake in this preparation and few reports investigating DAT–protein interactions have utilized this preparation. Following SDS-PAGE and colloidal Coomassie staining, selected protein bands from a DAT-immunoprecipitate were excised, digested with trypsin, extracted, and analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). From the analysis of the tryptic peptides, several proteins were identified including DAT, Ca 2+/calmodulin-dependent protein kinase II (CaMKII) β, CaMKII δ, protein kinase C (PKC) β, and PKC γ. Co-immunoprecipitation of PKC, CaMKII, and protein interacting with C kinase-1 with DAT was confirmed by Western blotting. Thus, the present study highlights a method to immunoprecipitate DAT and to identify co-immunoprecipitating proteins using LC/MS/MS and Western blotting. This method can be utilized to evaluate DAT protein–protein interactions but also to assess interactions involving other synaptic proteins. Ex vivo identification of protein–protein interactions will provide new insight into the function and regulation of a variety of synaptic, membrane-associated proteins, including DAT.

Neurochemical evidence that 3-methylglutaric acid inhibits synaptic Na+,K+-ATPase activity probably through oxidative damage in brain cortex of young rats

3-Methylglutaconic aciduria (MGTA) comprehends a group of disorders biochemically characterized by accumulation of 3-methylglutaric acid (MGA), 3-methylglutaconic acid (MGT) and occasionally 3-hydroxyisovaleric acid (OHIVA). Although neurological symptoms are common in the affected individuals, the mechanisms of brain damage are poorly known. In the present study we investigated the in vitro effect MGA, MGT and OHIVA, at concentrations ranging from 0.1 to 5.0mM, on bioenergetics and oxidative stress in synaptosomal preparations isolated from cerebral cortex of young rats. MGA significantly reduced mitochondrial redox potential (25%), as determined by resazurin reduction, and inhibited the activity of Na+,K+-ATPase (30%), whereas MGT and OHIVA did not modify these parameters. Moreover, the inhibitory effect elicited by MGA on Na+,K+-ATPase activity was totally prevented by co-incubation with the scavenging antioxidants creatine and melatonin, implying a role for reactive species in this effect. MGA also increased 2′,7′-dichlorofluorescein (DCFH) oxidation (30%), reinforcing that this organic acid induces reactive species production. The present data indicate that MGA compromises mitochondrial function, elicits reactive species production and inhibits the activity of a crucial enzyme implicated in neurotransmission. It is therefore presumed that these deleterious effects may play a role in the pathophysiology of the brain damage observed in patients affected by disorders in which MGA accumulates.

Impact of STZ‐induced hyperglycemia and insulin‐induced hypoglycemia in plasma amino acids and cortical synaptosomal neurotransmitters

In this work, we evaluated the effects of streptozotocin (STZ)-induced hyperglycemia and an acute episode of insulin-induced hypoglycemia in plasma amino acids and cortical neurotransmitters. For that purpose, we used citrate (vehicle)-treated Wistar rats, STZ-treated rats [i.p., 50 mg/kg body weight], and STZ-treated rats injected with insulin [s.c., dose adjusted with blood glucose levels] 1 h prior to sacrifice to induce an acute episode of hypoglycemia. Plasma was collected for determination of amino acids levels. In addition, cortical synaptosomal preparations were obtained and the total levels of neurotransmitters, levels of aspartate, glutamate, taurine, and GABA released by the action of KCl, iodoacetic acid (IAA), ouabain, and veratridine, membrane potential and ATP levels were evaluated. Compared with control rats, plasma from hypoglycemic rats presented increased levels of aspartate, glutamate, glutamine, and taurine whereas GABA levels were decreased in STZ and hypoglycemic rats. Similarly, glutamate and taurine levels were increased in hypoglycemic synaptosomes while GABA decreased in hypoglycemic and STZ-diabetic synaptosomes. The depolarizing agent KCl promoted an increase in aspartate, glutamate, and taurine release from hypoglycemic synaptosomes. The highest release of neurotransmitters occurred in the presence of veratridine and ouabain, two other depolarizing agents, in all groups of experimental animals. However, a higher release of glutamate was observed in the diabetic and hypoglycemic synaptosomes. No alterations were observed in synaptosomal membrane potential and ATP levels. These results show that in the presence of a metabolic insult a higher release of excitatory amino acids occurs, which may underlay the neuronal injury observed in type 1 diabetic patients under insulin therapy.

Conformation states of the neuronal porosome complex

Porosomes are the universal secretory machinery of the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to expel intravesicular contents to the environment during cell secretion. In neurons, 12- to 17-nm cup-shaped lipoprotein structures possessing a central plug are present at the presynaptic membrane, where 40-50 nm in diameter synaptic vesicles transiently dock and fuse to release neurotransmitters. The neuronal porosome complex has been isolated, its composition determined and it has been both structurally and functionally reconstituted in artificial lipid membranes. Earlier studies using AFM (atomic force microscopy), EM (electron microscopy), electron density and 3D contour mapping provide the structure and assembly of proteins within the neuronal porosome complex at the nanoscale level. A set of eight protein units lining the neuronal porosome cup is present, each connected via spoke-like elements to a central plug, hypothesized for the rapid opening and closing of the structure to the outside. In the present study, ultrahigh-resolution imaging of the presynaptic membrane of isolated synaptosome preparations demonstrate, for the first time, the presence of neuronal porosomes in both their open and close conformations. The results suggests that the central plug is retracted into the porosome cup in its open conformation and pushed outward to seal the porosome opening, supporting the hypothesis that it operates as the opening-closing device of the complex.

Deconstructing Antiobesity Compound Action: Requirement of Serotonin 5-HT2B Receptors for Dexfenfluramine Anorectic Effects

The now-banned anorectic molecule, dexfenfluramine, promotes serotonin release through a serotonin transporter-dependent mechanism, and it has been widely prescribed for the treatment of obesity. Previous studies have identified that 5-HT(2B) receptors have important roles in dexfenfluramine side effects, that is, pulmonary hypertension, plasma serotonin level regulation, and valvulopathy. We thus investigated a putative contribution of 5-HT(2B) receptors in dexfenfluramine-dependent feeding behavior in mice. Interestingly, the hypophagic response to dexfenfluramine (3-10 mg/kg) observed in wild-type mice (1-4 h) was eliminated in mice lacking 5-HT(2B) receptors (5-HT(2B)(-/-)). These findings were further validated by the lack of hypophagic response to dexfenfluramine in wild-type mice treated with RS127445, a highly selective and potent antagonist (pKi=8.22 ± 0.24). Using microdialysis, we observed that in 5-HT(2B)(-/-) awake mice, the dexfenfluramine-induced hypothalamic peak of serotonin release (1 h) was strongly reduced (fourfold) compared with wild type. Moreover, using hypothalamic synaptosomes, we established the serotonergic neuron autonomous properties of this effect: a strong serotonin release was observed upon dexfenfluramine stimulation of synaptosome preparation from wild type but not from mice lacking active 5-HT(2B) receptors. These findings strongly suggest that activation of presynaptic 5-HT(2B) receptors is a limiting step in the serotonin transporter dependent-releasing effect of dexfenfluramine, whereas other serotonin receptors act downstream with respect to feeding behavior.

Spinal α2-Adrenoceptor-mediated Analgesia in Neuropathic Pain Reflects Brain-derived Nerve Growth Factor and Changes in Spinal Cholinergic Neuronal Function

Spinal alpha2-adrenoceptor stimulation produces analgesia in neuropathic pain states, and this effect in animals is blocked by the inhibitors of brain-derived neurotrophic factor (BDNF) function. In rats, alpha2-adrenoceptor stimulation normally inhibits acetylcholine release, but it excites release after nerve injury. The authors examined the roles of BDNF and excitatory Gs-protein in this change. Male rats underwent L5-L6 spinal nerve ligation (SNL), and their lumbar spinal dorsal horns with or without spinal BDNF infusion were used for either synaptosome preparation for acetylcholine release or immunostaining for choline acetyltransferase. SNL did not alter spontaneous release from synaptosomes or choline acetyltransferase immunoreactivity in the spinal dorsal horn, but it reduced KCl-evoked acetylcholine release. Dexmedetomidine inhibited KCl-evoked acetylcholine release in synaptosomes from normal rats, but it excited KCl-evoked release in synaptosomes from SNL rats, and both effects were blocked by the alpha2-adrenoceptor antagonist idazoxan. Spinal infusion of an antibody to BDNF reduced choline acetyltransferase immunoreactivity in the spinal dorsal horn in both normal and SNL rats and abolished facilitation of KCl-evoked acetylcholine release by dexmedetomidine in SNL rats. Dexmedetomidine facilitation of acetylcholine release was also blocked by the inhibitors of Gs function. The increased reliance of spinal alpha2 adrenoceptors on cholinergic stimulation to cause analgesia after nerve injury reflects in part a shift from direct inhibition to direct excitation of spinal cholinergic neurons. The authors' results suggest that this shift relies on an interaction with Gs-proteins and BDNF.

Upregulation of the GABA transporter GAT-1 in the gracile nucleus in the spared nerve injury model of neuropathic pain

Neuropathic pain is a major health issue and is frequently accompanied by allodynia (painful sensations in response to normally non-painful stimulations), and unpleasant paresthesia/dysesthesia, pointing to alterations in sensory pathways normally dedicated to the processing of non-nociceptive information. Interestingly, mounting evidence indicate that central glial cells are key players in allodynia, partly due to changes in the astrocytic capacity to scavenge extracellular glutamate and γ-aminobutyric acid (GABA), through changes in their respective transporters (EAAT and GAT). In the present study, we investigated the glial changes occurring in the dorsal column nuclei, the major target of normally innocuous sensory information, in the rat spared nerve injury (SNI) model of neuropathic pain. We report that together with a robust microglial and astrocytic reaction in the ipsilateral gracile nucleus, the GABA transporter GAT-1 is upregulated with no change in GAT-3 or glutamate transporters. Furthermore, [ 3H] GABA reuptake on crude synaptosome preparation shows that transporter activity is functionally increased ipsilaterally in SNI rats. This GAT-1 upregulation appears evenly distributed in the gracile nucleus and colocalizes with astrocytic activation. Neither glial activation nor GAT-1 modulation was detected in the cuneate nucleus. Together, the present results point to GABA transport in the gracile nucleus as a putative therapeutic target against abnormal sensory perceptions related to neuropathic pain.

Comparison of a Protein-Level and Peptide-Level Labeling Strategy for Quantitative Proteomics of Synaptosomes Using Isobaric Tags

Quantitative proteomics using isobaric labeling typically involves sample digestion, peptide-level labeling and 2D LC-MS/MS. Proteomic analysis of complex samples can potentially be performed more comprehensively with GeLC-MS/MS. However, combining this approach with peptide-level labeling of multiple in-gel digests from entirely sectioned gel lanes can introduce many points of variation and adversely affect the final quantitative accuracy. Alternatively, samples labeled with isobaric tags at the protein level can be combined and analyzed by GeLC-MS/MS as a single gel lane. A caveat to this strategy is that only lysine residues are labeled, which might limit protein digestion and quantitation of peptides. Here we have compared a protein-level labeling GeLC-MS/MS strategy with a peptide-level labeling 2D LC-MS/MS approach, using mouse hippocampus synaptosomes and isobaric tandem mass tags. Protein-level labeling enabled the identification of 3 times more proteins (697 versus 241) than did peptide-level labeling, and importantly for quantitation, twice as many proteins with labeled peptides (480 versus 232) were identified. Preliminary in silico analysis also suggested the alternative use of Asp-N to trypsin to circumvent the interference of lysine labeling on protein digestion. Use of Asp-N resulted in the effective analysis of fewer peptides than with trypsin for the protein-level approach (1677 versus 3131), but yielded a similar quantitative proteomic coverage in terms of both peptides (1150 versus 1181) and proteins (448 versus 480). Taken together, these experiments demonstrate that protein-level labeling combined with GeLC-MS/MS is an effective strategy for the multiplexed quantitation of synaptosomal preparations, and may also be applicable to samples of a similar proteomic complexity and dynamic range of protein abundance.

The endogenous cannabinoid, anandamide, inhibits dopamine transporter function by a receptor‐independent mechanism

The endocannabinoid, anandamide (AEA), modulates the activity of the dopamine transporter (DAT) in heterologous cells and synaptosomal preparations. The cellular mechanisms mediating this effect are unknown. The present studies employed live cell imaging techniques and the fluorescent, high affinity DAT substrate, 4-(4-(dimethylamino)-styryl)-N-methylpyridinium (ASP(+)), to address this issue. AEA addition to EM4 cells expressing yellow fluorescent protein-tagged human DAT (hDAT) produced a concentration-dependent inhibition of ASP(+) accumulation (IC(50): 3.2 +/- 0.8 microM). This effect occurred within 1 min after AEA addition and persisted for 10 min thereafter. Pertussis toxin did not attenuate the effects of AEA suggesting a mechanism independent of G(i)/G(o) coupled receptors. The amidohydrolase inhibitor, phenylmethylsulfonyl fluoride (0.2 mM), failed to alter the AEA-evoked inhibition of ASP(+) accumulation. Methanandamide (10 microM), a metabolically stable analogue of AEA inhibited accumulation but arachidonic acid (10 microM) was without effect suggesting that the effects of AEA are not mediated by its metabolic products. The extent of AEA inhibition of ASP(+) accumulation was not altered in cells pre-treated with 1 microM URB597, a specific and potent fatty acid amide hydrolase inhibitor, and the cyclooxygenase inhibitor, indomethacin (5 microM) Live cell imaging revealed a significant redistribution of hDAT from the membrane to the cytosol in response to AEA treatment (10 microM; 10 min). Similarly biotinylation experiments revealed that the decrease in DAT function was associated with a reduction in hDAT cell surface expression. These results demonstrate that AEA modulates DAT function via a cannabinoid receptor-independent mechanism and suggest that AEA may produces this effect, in part, by modulating DAT trafficking.

Sexual steroids influence NTPDase3-expression in the neuroendocrine hypothalamus

Event Abstract Back to Event Sexual steroids influence NTPDase3-expression in the neuroendocrine hypothalamus Dávid S. Kiss1*, Virag Somogyi1, Andrea Gyorffy1, Tibor Bartha1, Sabrina Diano2, László V. Frenyó1 and Attila Zsarnovszky1 1 Szent Istvan University , Department of Physiology and Biochemistry, Faculty of Veterinary Sciences, Hungary 2 Yale University School of Medicine, Department of Obstetrics and Genecology, United States Introduction: We have previously determined the distribution pattern of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) in the rat brain and found that the hypothalamus is one of the brain regions richest in NTPDase3-immunoreactive (IR) neuronal structures. Next, we characterized the cellular and subcellular localization of NTPDase3-IR. Immunoprecipitate was found in distinct subcellular profiles, most importantly in mitochondria of hypothalamic excitatory neurons. Functional studies confirmed these findings. Since the neuroendocrine hypothalamus is the major target of gonadal steroid hormones, in the present study we aimed at determining the possible influence of estrogen and testosterone on the hypothalamic expression level and ATP-hydrolyzing enzymatic activity of NTPDase3. Methods: The medial and lateral subdivisions of the hypothalamus were investigated in castrated animals with or without hormone replacement and in intact controls. NTPDase3 protein levels were determined by Western blotting, and enzyme activity in neuronal mitochondria was investigated in synaptosomal preparations by the measurement of ADP-dependent state 3 mitochondrial respiration rates. Results and conclusion: Results revealed that both estrogen and testosterone exert a region-specific regulatory effect on hypothalamic NTPDase3-levels, and that these steroids differentially modulate the ATP-hydrolyzing activity of NTPDase in the medial- and lateral parts of the hypothalamus. Considering the previously proposed role of mitochondrial NTPDase3 in the regulation of neuronal ATP-levels, these results suggest that sexual steroids may regulate the energetic status of steroid-sensitive hypothalamic neurons. Conference: IBRO International Workshop 2010, Pécs, Hungary, 21 Jan - 23 Jan, 2010. Presentation Type: Poster Presentation Topic: Homeostatic and neuroendocrine systems Citation: Kiss DS, Somogyi V, Gyorffy A, Bartha T, Diano S, Frenyó LV and Zsarnovszky A (2010). Sexual steroids influence NTPDase3-expression in the neuroendocrine hypothalamus. Front. Neurosci. Conference Abstract: IBRO International Workshop 2010. doi: 10.3389/conf.fnins.2010.10.00099 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 23 Apr 2010; Published Online: 23 Apr 2010. * Correspondence: Dávid S Kiss, Szent Istvan University, Department of Physiology and Biochemistry, Faculty of Veterinary Sciences, Budapest, Hungary, Kiss.David@aotk.szie.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Dávid S Kiss Virag Somogyi Andrea Gyorffy Tibor Bartha Sabrina Diano László V Frenyó Attila Zsarnovszky Google Dávid S Kiss Virag Somogyi Andrea Gyorffy Tibor Bartha Sabrina Diano László V Frenyó Attila Zsarnovszky Google Scholar Dávid S Kiss Virag Somogyi Andrea Gyorffy Tibor Bartha Sabrina Diano László V Frenyó Attila Zsarnovszky PubMed Dávid S Kiss Virag Somogyi Andrea Gyorffy Tibor Bartha Sabrina Diano László V Frenyó Attila Zsarnovszky Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Ectonucleotidase and acetylcholinesterase activities in synaptosomes from the cerebral cortex of streptozotocin-induced diabetic rats and treated with resveratrol

The aim of the present study was to investigate the effects of resveratrol (RV), an important neuroprotective compound on NTPDase, 5′-nucleotidase and acetylcholinesterase (AChE) activities in cerebral cortex synaptosomes of streptozotocin (STZ)-induced diabetic rats. The animals were divided into six groups ( n = 8): control/saline; control/RV 10 mg/kg; control/RV 20 mg/kg; diabetic/saline; diabetic/RV 10 mg/kg; diabetic/RV 20 mg/kg. After 30 days of treatment with resveratrol the animals were sacrificed and the cerebral cortex was removed for synaptosomes preparation and enzymatic assays. The results demonstrated that NTPDase and 5′-nucleotidase activities were significantly increased in the diabetic/saline group ( p < 0.05) compared to control/saline group. Treatment with resveratrol significantly increased NTPDase, 5′-nucleotidase activities in the diabetic/RV10 and diabetic/RV20 groups ( p < 0.05) compared to diabetic/saline group. When resveratrol was administered per se there was also an increase in the activities of these enzymes in the control/RV10 and control/RV20 groups ( p < 0.05) compared to control/saline group. AChE activity was significantly increased in the diabetic/saline group ( p < 0.05) compared to control/saline group. The treatment with resveratrol prevented this increase in the diabetic/RV10 and diabetic/RV20 groups. In conclusion, this study demonstrated that the resveratrol interfere with the purinergic and cholinergic neurotransmission by altering NTPDase, 5′-nucleotidase and AChE activities in cerebral cortex synaptosomes of diabetic rats. In this context, we can suggest that resveratrol should be considered potential therapeutics and scientific tools to be investigated in brain disorders associated with the diabetes.

Abnormality of synaptic vesicular associated proteins in cerebral cortex and hippocampus after microwave exposure

Studies were performed to determine the effects of microwave on synaptic vesicles and the expression of synaptic vesicular associated proteins including synapsin I, VAMP-2, syntaxin, and synaptophysin. 25 Wistar rats were exposed to microwave which the average power density was 30 mW/cm(2), and whole body average specific absorption rate was 14.1 W/kg for 5 min. Synaptosome preparations in the cerebral cortex and hippocampus were obtained by isotonic Percoll/sucrose discontinuous gradients at 6 h, 1, 3, and 7 days after radiation. The expression of synaptic vesicular associated proteins was measured using Western blots and image analysis. The interaction between VAMP-2 and syntaxin was examined by coimmunoprecipitation analysis. Synapsin I in the cerebral cortex were decreased at 3 days (P < 0.01) after radiation and in the hippocampus increased at 1 day (P < 0.01), decreased at 3 days (P < 0.01), increased again at 7 days (P < 0.01) after exposure, compared with the sham-treated controls. Synaptophysin were increased in 1-7 days (P < 0.01) after exposure in the cerebral cortex and hippocampus. VAMP-2 were decreased at 1 and 3 days (P < 0.01) and syntaxin were decreased in 6 h to 3 days (P < 0.01) after radiation in the cerebral cortex and hippocampus. The interactions between VAMP-2 and syntaxin were decreased at 3-7 days (P < 0.01) after radiation in the cerebral cortex and hippocampus, compared with the sham-treated controls. These results suggest that 30 mW/cm(2) (SAR 14.1 W/kg) microwave radiation can result in the perturbation of the synaptic vesicles associated proteins: synapsin I, synaptophysin, VAMP-2, and syntaxin. The perturbation could induce the deposit of synaptic vesicle, which might be relative to the dysfunction of the synaptic transmission, even the cognition deficit.

Hypericum perforatum L. (St John's wort) extract Ze 117 inhibits dopamine re‐uptake in rat striatal brain slices. An implication for use in smoking cessation treatment?

Classic synthetic antidepressant drugs, as well as St John's wort extract (SJW), directly inhibit the re-uptake of norepinephrine (NE) and/or serotonin (5-HT) into pre-synaptic axons. With chronic treatment they induce adaptive changes in a number of neurotransmitter receptors in synaptic membranes. The immediate effects of SJW Ze 117, an extract low in hyperforin content, on the specific dopamine (DA) uptake were studied in rat striatal brain slices and compared with the effects on NE and 5-HT uptake in rat cortical brain slices. Specific DA uptake was inhibited in a dose dependent manner. In contrast to the findings in synaptosomal preparations published so far, the extract showed different inhibitory potencies for the respective transporters. The potencies for the uptake inhibition of NA, DA and 5-HT were 30, 7 and 1, respectively. The results indicate that the SJW Ze 117 extract interferes in three ways with the individual uptakes of the relevant neurotransmitters that are considered to be causal in the development of depression. This observation, the concomitant and potent inhibition of DA re-uptake by SJW extract, may additionally provide a rationale for the treatment of nicotine or drug addiction with SJW.

Molecular Requirements for Recognition of Brain Voltage-gated Sodium Channels by Scorpion α-Toxins

The scorpion alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus) is active at various mammalian voltage-gated sodium channels (Na(v)s) and is inactive at insect Na(v)s. To resolve the molecular basis of this preference we used the following strategy: 1) Lqh2 was expressed in recombinant form and key residues important for activity at the rat brain channel rNa(v)1.2a were identified by mutagenesis. These residues form a bipartite functional surface made of a conserved "core domain" (residues of the loops connecting the secondary structure elements of the molecule core), and a variable "NC domain" (five-residue turn and the C-tail) as was reported for other scorpion alpha-toxins. 2) The functional role of the two domains was validated by their stepwise construction on the similar scaffold of the anti-insect toxin LqhalphaIT. Analysis of the activity of the intermediate constructs highlighted the critical role of Phe(15) of the core domain in toxin potency at rNa(v)1.2a, and has suggested that the shape of the NC-domain is important for toxin efficacy. 3) Based on these findings and by comparison with other scorpion alpha-toxins we were able to eliminate the activity of Lqh2 at rNa(v)1.4 (skeletal muscle), hNa(v)1.5 (cardiac), and rNa(v)1.6 channels, with no hindrance of its activity at Na(v)1.1-1.3. These results suggest that by employing a similar approach the design of further target-selective sodium channel modifiers is imminent.

NTPDases in the neuroendocrine hypothalamus: possible energy regulators of the positive gonadotrophin feedback.

BackgroundBrain-derived ectonucleoside triphosphate diphosphohydrolases (NTPDases) have been known as plasma membrane-incorporated enzymes with their ATP-hydrolyzing domain outside of the cell. As such, these enzymes are thought to regulate purinergic intercellular signaling by hydrolyzing ATP to ADP-AMP, thus regulating the availability of specific ligands for various P2X and P2Y purinergic receptors. The role of NTPDases in the central nervous system is little understood. The two major reasons are the insufficient knowledge of the precise localization of these enzymes in neural structures, and the lack of specific inhibitors for the various NTPDases. To fill these gaps, we recently studied the presence of neuron-specific NTPDase3 in the mitochondria of hypothalamic excitatory neurons by morphological and functional methods. Results from those studies suggested that intramitochondrial regulation of ATP levels may play a permissive role in the neural regulation of physiological functions by tuning the level of ATP-carried energy that is needed for neuronal functions, such as neurotransmission and/or intracellular signaling.Presentation of the hypothesisIn the lack of highly specific inhibitors, the determination of the precise function and role of NTPDases is hardly feasable. Yet, here we attempt to find an approach to investigate a possible role for hypothalamic NTPDase3 in the initiation of the midcycle luteinizing hormone (LH) surge, as such a biological role was implied by our recent findings. Here we hypothesize that NTPDase-activity in neurons of the AN may play a permissive role in the regulation of the estrogen-induced pituitary LH-surge.Testing the hypothesisWe propose to test our hypothesis on ovariectomized rats, by stereotaxically injecting 17beta-estradiol and/or an NTPDase-inhibitor into the arcuate nucleus and determine the consequential levels of blood LH, mitochondrial respiration rates from arcuate nucleus synaptosomal preparations, NTPDase3-expression from arcuate nucleus tissue samples, all compared to sham and intact controls.Implications of the hypothesisResults from these studies may lead to the conclusion that estrogen may modulate the activity of mitochondrial, synapse-linked NTPDase3, and may show a correlation between mitochondrial NTPDase3-activity and the regulation of LH-release by estrogen.

Synaptic proteome changes in the superior frontal gyrus and occipital cortex of the alcoholic brain

Cognitive deficits and behavioral changes that result from chronic alcohol abuse are a consequence of neuropathological changes which alter signal transmission through the neural network. To focus on the changes that occur at the point of connection between the neural network cells, synaptosomal preparations from post-mortem human brain of six chronic alcoholics and six non-alcoholic controls were compared using 2D-DIGE. Functionally affected and spared regions (superior frontal gyrus, SFG, and occipital cortex, OC, respectively) were analyzed from both groups to further investigate the specific pathological response that alcoholism has on the brain. Forty-nine proteins were differentially regulated between the SFG of alcoholics and the SFG of controls and 94 proteins were regulated in the OC with an overlap of 23 proteins. Additionally, the SFG was compared to the OC within each group (alcoholics or controls) to identify region specific differences. A selection were identified by MALDI-TOF mass spectrometry revealing proteins involved in vesicle transport, metabolism, folding and trafficking, and signal transduction, all of which have the potential to influence synaptic activity. A number of proteins identified in this study have been previously related to alcoholism; however, the focus on synaptic proteins has also uncovered novel alcoholism-affected proteins. Further exploration of these proteins will illuminate the mechanisms altering synaptic plasticity, and thus neuronal signaling and response, in the alcoholic brain.

Synthesis of new β- and γ-benzyloxy- S-glutamic acid derivatives and evaluation of their activity as inhibitors of excitatory amino acid transporters

An efficient stereoselective preparation of the two enantiomerically pure diasteroisomers of γ-benzyloxy- S-glutamic acid was performed using trans-4-hydroxy- l-proline as a source of chirality, while the erythro and threo isomers of β-benzyloxy- S-glutamic acid were prepared starting from ( R)-Garner's aldehyde. All new derivatives were tested for their inhibitory activity against excitatory amino acid transporters in a rat synaptosomal preparation and their IC 50 values were compared to that of TBOA, a one carbon lower homologue commonly used as the reference blocker of glutamate transporters.

A Receptor Mechanism for Methamphetamine Action in Dopamine Transporter Regulation in Brain

This study reveals a novel receptor mechanism for methamphetamine action in dopamine transporter (DAT) regulation. Trace amine-associated receptor 1 (TAAR1) is expressed in brain dopaminergic nuclei and is activated by methamphetamine in vitro. Here, we show that methamphetamine interaction with TAAR1 inhibits [(3)H]dopamine uptake, enhances or induces [(3)H]dopamine efflux, and triggers DAT internalization. In time course assays in which methamphetamine and [(3)H]dopamine were concurrently loaded into cells or synaptosomes or in pretreatment assays in which methamphetamine was washed away before [(3)H]dopamine loading, methamphetamine caused a distinct inhibition in [(3)H]dopamine uptake in TAAR1 + DAT-cotransfected cells and in wild-type mouse and rhesus monkey striatal synaptosomes. This distinct uptake inhibition was not observed in DAT-only transfected cells or in TAAR1 knockout mouse striatal synaptosomes. In [(3)H]dopamine efflux assays using the same cell and synaptosome preparations, methamphetamine enhanced [(3)H]dopamine efflux at a high loading concentration of [(3)H]dopamine (1 muM) or induced [(3)H]dopamine efflux at a low loading concentration of [(3)H]dopamine (10 nM) in a TAAR1-dependent manner. In DAT biotinylation assays using the same cell and synaptosome preparations, we observed that 1 muM methamphetamine induced DAT internalization in a TAAR1-dependent manner. All these TAAR1-mediated effects of methamphetamine were blocked by the protein kinase inhibitors H89 [N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline] and/or 2-{8-[(dimethylamino) methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl}-3-(1-methylindol-3-yl)maleimide (Ro32-0432), suggesting that methamphetamine interaction with TAAR1 triggers cellular phosphorylation cascades and leads to the observed effects of methamphetamine on DAT. These findings demonstrate a mediatory role of TAAR1 in methamphetamine action in DAT regulation and implicate this receptor as a potential target of therapeutics drugs for methamphetamine addiction.

Mechanism of CCK‐induced oxytocin release from hypothalamic synaptosomal preparation

Previously we have reported that both KCl and CCK‐8 cause oxytocin release from spinal cord synaptosomes. We have also demonstrated the involvement of mitochondria in this release process. Mitochondria could modulate OT release either as a source of Ca2+ or as a source of ATP or both. The current experiments were designed to determine if ATP has some modulatory role in this release mechanism. Present experiments were conducted with hypothalamic synaptosomes prepared from hypothalamic block obtainedfrom freshly sacrificed rats containing both PVN and SON. Synaptosomes were purified by discontinuous percoll gradient (23%,15%,10% &amp;3%). The interface between 23% &amp;15 % was collected and used for the release study. For the current experiments ATP was used as a stimulant. ATP in a concentration of 10nM, 50 nM and 100 nM caused release of oxytocin 90%,364% &amp; 77% rtespectively.. Thus, these results suggest that inhibition of oxytocin release from spinal cord synaptosomes following treatment with electron transport chain inhibitors was probably due to lack of ATP and not due to lack of Ca2+

The PKC inhibitor Ro31-8220 blocks acute amphetamine-induced dopamine overflow in the nucleus accumbens

Acute administration of the psychostimulant amphetamine increases extracellular levels of dopamine (DA) by reversing the DA transporter on ascending midbrain DA neurons. In vitro studies using striatal synaptosomal, slice and nucleus accumbens (NAcc) tissue preparations have implicated protein kinase C (PKC) in this effect. The present study further examined this effect in vivo by assessing the ability of the PKC inhibitor, Ro31-8220 (10 μM), to inhibit acute amphetamine-induced DA overflow when applied with this drug to the NAcc via reverse dialysis. Amphetamine was applied at a concentration of 30 μM, and the core and shell subregions of the NAcc were assayed separately in freely moving rats. These brain regions play a role in the acute locomotor-activating and motivational effects of amphetamine. Consistent with the findings of previous in vitro experiments, reverse dialysis of Ro31-8220 with amphetamine robustly attenuated the ability of this drug to increase extracellular levels of dopamine in both the core and shell subregions of the NAcc. These results confirm that amphetamine stimulates dopamine overflow via a PKC-dependent mechanism.